Method and apparatus for detecting low light levels

ABSTRACT

A method and apparatus for measuring very low light signals including integrating a signal from a photo diode, avalanche photo diode, photomultiplier tube or the like, digitally sampling the integrator output more than two times during each integration period, fitting a curve to the multiple digitized readings to calculate the integration slope for each integration period and determining the original signal from the calculated integration slope.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/104,813, filed Jun. 25, 1998, which is a continuation of U.S. patentapplication Ser. No. 60/051,102, filed Jun. 27, 1997, the disclosure ofwhich is hereby incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

This application relates in general to measurement and sensing of lowpower signals. More particularly, the invention relates to the sensing,amplification and measurement of a low power, light-based signal.

FIG. 1 illustrates a circuit 100 of the prior art for amplifying asignal from a photo diode 130. The circuit of FIG. 1 includes the photodiode 130 connected across the inputs of an operational amplifier 120.The positive input of the op amp 120 is tied to ground. A resistive loadR 150 is coupled between the negative terminal and the out signal 110 ofthe op amp 120.

Notably, the feedback resistor R 150 has inherent thermal noise that cansometimes exceed the actual signal from the photo diode 130. The outputfrom a resistive feedback amplifier such as circuit 100 is given inequation (1) below:

    V.sub.out =-i R                                            1

where V_(out) is in volts, i is the input signal in amperes from asignal source (such as photo diode 130) and R is the feedback resistance(such as the resistor R 150) in ohms.

A component with resistance generates thermal noise with the followingRMS values: ##EQU1## where V_(RMS) noise is in volts and I_(RMS) noiseis in amperes and where k=1.38×10⁻²³ J/° K (Boltzmann's constant), T isthe absolute temperature in ° K, B is the bandwidth in Hz and R is theresistance in ohms.

Therefore, when an application requires the amplification of a very lowsignal from a photo diode, the prior art resistive feedback amplifier100 sometimes proves unuseful due to excessive noise, for example.

FIG. 2 presents a circuit 200 of the art, designed to avoid this thermalnoise problem. In FIG. 2, the photo diode 130 remains coupled across theinputs of the op amp 120. In place of the resistive element R 150, acapacitor 220, coupled between the negative input and the output 210 ofthe op amp 120, serves as the feedback element. The source of afield-effect transistor (FET) 230 is coupled to the output 210 of the opamp 120 while the drain is coupled to the negative input of the op amp120. The gate of the FET 230 serves as a Reset signal 240.

The use of the capacitor 220 as the feedback element eliminates thenoise problem of the circuit 100.

The output from an integrator such as the circuit 200 is given inequation (4) below: ##EQU2## where i is the input signal from a signalsource (such as photo diode 130) in amperes, t is the time from reset toreading in seconds and C is the feedback capacitance (of capacitor 220,for example) in farads.

FIG. 3 illustrates the timing of the operation of the circuit 200 ofFIG. 2. A control circuit (not shown) typically resets the integrator200 (by means of the Reset signal 240) at twice the rate of the signalbandwidth. Just prior to each of these resets, the control circuit readsthe out signal 210 and extracts the true signal.

The use of the semiconductor switch 230, however, creates its ownproblems in the circuit 200. The charge transfer itself from the Resetsignal 240 during the resetting of the integrator 200 induces noise. Toavoid this problem, the control circuit reads the out signal 210 rightafter releasing the reset switch 240. The control circuit then subtractsthis reading from the final reading.

The noise of the photo diode 130 and op amp 120 nonetheless affect thetwo-reading scheme used with the circuit 200 up to the bandwidth of thesystem. The system bandwidth has to be much higher than the signalbandwidth in order not to distort the integration curves.

Accordingly, there is a need for a circuit for an improved detector oflow levels of light without the thermal noise and other problemsdescribed above. These and other goals of the invention will be readilyapparent to one of ordinary skill in the art on the reading of thebackground above and the invention description below.

SUMMARY OF THE INVENTION

Herein is disclosed a method and apparatus for measuring very low powersignals such as low power light signals, including integrating a signalfrom a signal source such as a photo diode, an avalanche photo diode, aphotomultiplier tube or the like, digitally sampling the integratoroutput multiple times during each integration period, fitting a curve tothe multiple digitized readings to calculate the integration slope foreach integration period and determining the original signal from thecalculated integration slope.

According to an aspect of the invention, an apparatus for use inmeasuring low power signals is provided, the apparatus comprising: anintegrator, wherein the integrator receives an original low power signalfrom a signal source and integrates the signal over multiple integrationperiods; an analog-to-digital converter having an analog input coupledto an output of the integrator, wherein the converter digitally samplesthe integrator output more than two times during each integration periodto obtain multiple digital samples; and a processor coupled to a digitaloutput of the analog-to-digital converter, wherein the processordetermines the original low power signal from the multiple digitalsamples.

According to another aspect of the invention, an apparatus for use inmeasuring low power light-based signals in a detection region in a firstone of at least two intersecting microchannels is provided, theapparatus comprising: a photo diode located proximal the detectionregion which detects a low power light-based signal in the detectionregion and outputs a photo diode signal; an integrator having an inputcoupled to an output of the photo diode, wherein the integrator receivesand integrates the photo diode signal over multiple integration periods;a low pass filter having an input coupled to an output of theintegrator, wherein the low pass filter operates to filter outfrequencies above a predetermined level in the integrator output signal;an analog-to-digital converter having an analog input coupled to anoutput of the low pass filter, wherein the converter digitally samplesthe filtered integrator output signal more than two times during eachintegration period to obtain multiple digital samples; and a processorcoupled to a digital output of the analog-to-digital converter, whereinthe processor calculates the integration slope for each integrationperiod using the multiple digital samples, and wherein the processordetermines the original low power signal from the calculated integrationslopes.

According to yet another aspect of the invention, a method is providedfor measuring low power signals, the method comprising the steps of:receiving an original signal from a signal source; integrating overmultiple integration periods the original signal with an integrator toproduce an integrator output signal; digitally sampling the integratoroutput signal more than two times during each integration period with ananalog-to-digital converter coupled to the integrator to obtain multipledigital samples; and determining the original signal from the multipledigital samples.

According to a further aspect of the invention, a method is provided formeasuring low power light-based signals in a detection region in a firstone of at least two intersecting microchannels, the method comprisingthe steps of: locating a photo diode proximal the detection region,wherein the photo diode detects an original low power light-based signalin the detection region and outputs a photo diode signal; integratingthe photo diode signal over multiple integration periods to produce anintegrator output signal using an integrator having an input coupled toan output of the photo diode; filtering out frequencies above apredetermined level in the integrator output signal using a low passfilter having an input coupled to an output of the integrator; digitallysampling the filtered integrator output signal more than two timesduring each integration period with an analog-to-digital converterhaving an analog input coupled to an output of the low pass filter toobtain multiple digital samples; calculating the integration slope foreach integration period using the multiple digital samples; anddetermining the original low power signal from the calculatedintegration slopes.

According to yet a further aspect of the invention, a system is providedfor measuring low power signals, the system comprising: means fordetecting an original low power signal; means for integrating theoriginal low power signal over multiple integration intervals to producean integration output signal; digital sampling means for digitallysampling the integration output signal more than two times during eachintegration interval to obtain multiple digital samples; and a processorcoupled to the digital sampling means, the processor including: meansfor calculating the integration slope for each integration intervalusing the multiple digital samples; and means for determining theoriginal low power signal from the calculated integration slopes.

Reference to the remaining portions of the specification, including thedrawings and claims, will realize other features and advantages of thepresent invention. Further features and advantages of the presentinvention, as well as the structure and operation of various embodimentsof the present invention, are described in detail below with respect tothe accompanying drawings. In the drawings, like reference numbersindicate identical or functionally similar elements.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a circuit of the prior art for amplifying a signalfrom a photo diode;

FIG. 2 presents a circuit of the prior art, designed to avoid thethermal noise problem;

FIG. 3 illustrates the timing of the operation of the circuit of FIG. 2;

FIG. 4 illustrates a circuit 400 according to the invention;

FIG. 5 illustrates the timing of the operation of the circuit 400 ofFIG. 4;

FIG. 6 illustrates the overall operation of the circuit of FIG. 4; and

FIG. 7 illustrates an example of a microfluidic device for use withcertain aspects of the present invention.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

In preferred aspects, the method and apparatus of the instant inventionare used in the detection of light-based signals from analytical systemsemploying optical detection in microscale fluidic channels. Examplesinclude, e.g., fused silica capillary systems, i.e., CE, as well asmicrofluidic devices and systems that incorporate microscale channelssuch as microfluidic channels. Such systems are generally described inU.S. patent application Ser. Nos. 08/845,754 (Attorney Docket No.100/01000, filed Apr. 25, 1997), 08/881,696 (Attorney Docket No.17646-000420, filed Jun. 24, 1997), a continuation-in-part of U.S.patent application Ser. No. 08/761,575 (filed Dec. 6, 1996), and60/049,013 (Attorney Docket No. 17646-003600), filed Jun. 9, 1997. (Thedisclosure of each of these applications is hereby incorporated byreference in its entirety for all purposes.)

A "microfluidic" channel is a channel (groove, depression, tube, etc.)which is adapted to handle small volumes of fluid. In a typicalembodiment, the channel is a tube, channel or conduit having at leastone subsection with at least one cross-sectional dimension of betweenabout 0.1 μm and 500 μm, and typically less than 100 μm; ordinarily, thechannel is closed over a significant portion of its length, having top,bottom and side surfaces. In operation, materials that are beinganalyzed, e.g., subjected to optical analysis for light based signals,in these microscale fluidic systems, are transported along themicroscale fluid channels, past a detection point, where a detectablesignal indicative of the presence or absence of some material orcondition, is measured. In the case of light based detection systems,the signals within these channels typically result from the presence oflight emitting substances therein, e.g., fluorescent or chemiluminescentmaterials, that are used as indicators of the presence of absence ofsome material or condition. Because microscale channels have extremelysmall dimensions, the amount of signal typically available for detectionwithin such channels is also extremely small. For example, in themicrofluidic systems for which the present invention is particularlyuseful, the power levels of signals from a detection region in amicrofluidic channel are typically on the order of about 0.1 pW to about10 pW.

As noted above, in microscale analytical systems, a signal bearingmaterial is transported along the microscale channel and past adetection point. Typically, transporting materials within these systemsmay be carried out by any of a variety of methods. For example, suchmaterial transport is optionally carried out through the application ofpressures to the materials within the channels, through theincorporation of microscale mechanical pumps, or through the applicationof electric fields, to move materials through the channels.

In preferred aspects, the above microfluidic systems use electrokinetictransport systems for moving material within the microfluidic channels.As used herein, "electrokinetic material transport systems" includesystems which transport and direct materials within an interconnectedchannel and/or chamber containing structure, through the application ofelectrical fields to the materials, thereby causing material movementthrough and among the channel and/or chambers (i.e., cations will movetoward the negative electrode, while anions will move toward thepositive electrode). Such electrokinetic material transport anddirection systems include those systems that rely upon theelectrophoretic mobility of charged species within the electric fieldapplied to the structure. Such systems are more particularly referred toas electrophoretic material transport systems. Other electrokineticmaterial direction and transport systems rely upon the electroosmoticflow of fluid and material within a channel or chamber structure whichresults from the application of an electric field across suchstructures. In brief, when a fluid is placed into a channel which has asurface bearing charged functional groups, e.g., hydroxyl groups inetched glass channels or glass microcapillaries, those groups canionize. In the case of hydroxyl functional groups, this ionization(e.g., at neutral pH), results in the release of protons from thesurface and into the fluid, creating a concentration of protons at nearthe fluid/surface interface, or a positively charged sheath surroundingthe bulk fluid in the channel. Application of a voltage gradient acrossthe length of the channel will cause the proton sheath to move in thedirection of the voltage drop (i.e., toward the negative electrode).

FIG. 7 depicts an example of a microfluidic device for use with certainaspects of the present invention. As shown, the device 300 includes abody structure 302 which has an integrated channel network 304 disposedtherein. The body structure 302 includes a plurality of reservoirs306-328, disposed therein, for holding reagents, sample materials, andthe like. Also included is buffer reservoir 330, as well as wastereservoirs 332, 334 and 336. The reagents, samples, etc. are transportedfrom their respective reservoirs, either separately or together withother reagents from other reservoirs into a main channel 338, and alongmain channel 338 toward waste reservoir 336, past detection zone orwindow 340. Detection window 340 is typically transparent, and may becomprised of a transparent region of the body structure, or a separatetransparent window fabricated into the body structure. Typically, thebody structure is itself fabricated from a transparent material, e.g.,glass or transparent polymers, thereby obviating the need for a separatetransparent region to define the detection window. Microfluidic devicesof the sort described above are useful in performing a variety ofanalyses, such as electrophoretic separation of macromolecules, e.g.,nucleic acids, proteins, etc. (see U.S. application Ser. No. 08/845,754,filed Apr. 25, 1997, and previously incorporated herein by reference),high throughput screening assays, e.g., in pharmaceutical discovery, anddiagnostics, e.g., immunoassays (see, e.g., Published InternationalApplication No. WO98/00231).

In one embodiment, a signal source is located proximal detection window340 for detecting low power, light-based signals from the detectionregion. The signal source is optionally selected from a number ofdifferent types of light detectors, i.e., photo diodes, avalanche photodiodes, photomultiplier tubes (PMTs) and the like. In preferred aspects,a photo diode is used. FIG. 4 illustrates a circuit 400 for amplifying asignal from a photo diode 130 according to the invention. In FIG. 4, thephoto diode 130 is coupled across the inputs of an op amp 120. Acapacitor 220, coupled between the negative input and the output 210 ofthe op amp 120, serves as the feedback element. The source of afield-effect transistor (FET) 230 is coupled to the output 210 of the opamp 120 while the drain is coupled to the negative input of the op amp120. The gate of the FET 230 is connected to Reset signal 440.

The input of a low-pass filter 410 is coupled to the output signal 210.The output of the low-pass filter 410 is coupled to the analog input ofan analog-to-digital converter 420. Finally, a microprocessor 430receives as input the digitized output signal 450 of theanalog-to-digital converter 420.

FIG. 6 illustrates the overall operation of the circuit 400. The circuit400 receives and integrates a signal from a photo diode and resets theintegrator, step 610. The circuit 400 then filters out the higherfrequencies in the integrated signal, step 620. Next, the circuit 400converts the analog filtered and integrated signal to digital samples,step 630. Finally, the circuit 400 calculates the integration slope forthe photo diode signal by fitting a curve to the digitized samples, step640. With the calculated slopes, the circuit 400 is better able todetermine the original noise-less signal from the photo diode.

FIG. 5 illustrates the timing of operation of the circuit 400 of FIG. 4.In contrast to the prior art circuit 200 which just takes two readingsfor each integration period, the circuit 400 takes many readings 530 foreach integration period. In preferred aspects, analog-to-digitalconverter 420 samples the integrator output more than two times,preferably more than ten times, still more preferably more thanone-hundred times, in many cases more that five hundred and even morethan one-thousand times.

Also in contrast to the prior art circuit 200, the circuit 400 applies amore sophisticated curve-calculation routine to the per-period samplereadings to generate the per-period calculated slopes 510 and 520. Thecurve calculation filters away overlying noise. In this way, the circuit400 decreases the noise contribution from the photo diode 130 and the opamp 120 near to what is included in the signal bandwidth.

The frequency of the Reset signal 440, f_(Reset), is fast enough toallow detection of the fastest signal necessary, f_(Signal). In oneembodiment, f_(Reset) is approximately twice f_(Signal).

The frequency of the low-pass filter 410, f_(Low-Pass), is fast enoughthat the integration curves do not become significantly disturbed.f_(Low-Pass) is dependent on signal distortion specifications. In oneembodiment, f_(Low-pass) is approximately ten times f_(Reset).

To best filter noise, in one embodiment, the sample frequency,f_(sample), is at least twice f_(Low-pass).

The microprocessor 430 uses any of the numerous curve-fitting algorithmsknown in the art to calculate the slope of each integration period.Least-squares curve fitting is but one example of these algorithms. Anycurve-fitting algorithm that filters away overlaid noise can be used.For example, the curve-fitting algorithm can be: 0.5×f_(Reset) ×(FirstReadings-Last Readings), where "First Readings" are the first half ofthe samples taken within an integration period and "Last Readings" arethe second half of the samples taken within an integration period.

The circuit 400 filters both voltage noise and current noise from the opamp 120 and photo diode 130 close to the theoretical value included inthe signal band. Noise can be almost totally ignored.

The noise in the measurement is affected by the amount of noise at thenegative input of the integrating op amp 120. Any component willgenerate noise as described in equations (2) and (3) above. Therefore,in a preferred embodiment, all components connected to the negativeinput of op amp 120 have very high resistance. Also, op amp 120preferably has low noise parameters.

The embodiments described herein are by way of example and notlimitation. Modifications to the invention as described will be readilyapparent to one of ordinary skill in the art. For example, while thephoto diode 130 is described as a signal source above, it is understoodthat any sensor giving voltage or current signals or any source ofreadings convertible to current or voltage readings can be the signalsource. (Of course, if the signal source is a voltage output, a resistorconverts it to a current output adaptable to the circuit described.)Still further, while a FET device 230 is described as the resettingmechanism, other devices which have high resistance when not assertingthe Reset signal 440 can be used. For example, the resetting mechanismcan be an opto-activated FET or opto-activated diode or relay or anotherkind of transistor.

Of course, the program text for such software as is herein disclosed canexist in its static form on a magnetic, optical or other disk, onmagnetic tape or other medium requiring media movement for storageand/or retrieval, in ROM, in RAM or other integrated circuit, or inanother data storage medium. That data storage medium may be integral toor insertable into a computer system.

What is claimed is:
 1. In a signal met system having a signal processor,including means for integrating a signal and means for sampling asignal, a computer program code contained in a fixed computer readablemedium that when loaded into the signal processor and run will cause thesystem to measure low power signals by performing the stepsof:integrating over multiple integration periods an original signalreceived from a signal source with the integration means to produce anintegrated signal; digitally sampling the integrated signal multipletimes during each integration period of the integrated signal with thesampling means to obtain multiple digital samples; and determining theoriginal signal from the multiple digital samples.
 2. The computerprogram code of claim 1, wherein when loaded into the signal processorand run further causes the system to filter out frequencies above apredetermined level in the integrator signal to produce a filteredsignal, wherein the filtered signal is digitally sampled by the samplingmeans.
 3. The computer program code of claim 1, wherein when loaded intothe signal processor and run further causes the system to generate areset signal, wherein the reset signal triggers the beginning of eachintegration period.
 4. The computer program code of claim 1, whereinwhen loaded into the signal processor and run further causes the systemto determine the original signal from the multiple digital samples bycalculating the integration slope for each integration period using themultiple digital samples, and determining the original signal from thecalculated integration slopes.
 5. In a signal measuring system having asignal processor, including means for integrating a signal and means forsampling a signal, a computer program code contained in a fixed computerreadable medium that when loaded into the signal processor and run willcause the system to measure low power signals by performing the stepsof:integrating over multiple integration periods an original signalreceived from a signal source with the integration means to produce anintegrated signal; digitally sampling the integrated signal multipletimes during each integration period of the integrated signal with thesampling means to obtain multiple digital samples; calculating theintegration slope for each integration period using the multiple digitalsamples; and determining the original signal from the calculatedintegration slopes.
 6. In a signal measuring system having a signalprocessor, including means for integrating a signal and means forsampling a signal, a computer program code contained in a fixed computerreadable medium that when loaded into the signal processor and run willcause the system to measure low power light-based signals in a detectionregion in a first one of at least two intersecting microchannels byperforming the steps of:integrating a first signal received from a photodiode over multiple integration periods to produce an integrated signalusing the integration means, wherein the photo diode detects an originallow power light-based signal in the detection region and outputs thefirst signal; filtering out frequencies above a selected level in theintegrated signal to produce a filtered signal; digitally sampling thefiltered signal multiple times during each integration period with thesampling means to obtain multiple digital samples; calculating theintegration slope for each integration period using the multiple digitalsamples; and determining the original low power signal from thecalculated integration slopes.